The discovery that RNA can catalyze chemical reactions revealed a novel role for these molecules that extends beyond the transfer of genetic information. To date, only a small number of biologically occurring catalytic RNAs (ribozymes) have been identified. This may be a result of the absence of unbiased, activity- based methods for their identification. Approaches that do not rely on secondary structure prediction and instead select RNAs possessing enhanced reactivity could reveal novel ribozymes. This proposal describes the development of a chemical approach to the discovery of biological ribozymes. This activity-based approach employs small-molecule probes designed to covalently label RNAs possessing enhanced chemical reactivity. The strategy for the design of ribozyme-specific chemical probes relies on the well-validated assumption that ribozymes employ similar tools as protein catalysts; the tertiary structures of most ribozymes result in functional groups with enhanced nucleophilicity that are largely responsible for catalysis. Appropriately designed electrophilic compounds (activity-based probes) designed to selectively label highly nucleophilic RNAs will be equipped with an affinity tag (biotin) and applied to the selection of unusually reactive RNAs from RNA pools derived from the genomes of a range of bacterial and eukaryotic organisms. The chemical reactivity and biological roles of any newly identified RNAs will be fully characterized.